The pathological isoform of the prion protein (PrPres) can serve as a marker for prion diseases, but more practical tests are needed for preclinical diagnosis and sensitive detection of many prion infections. Previously we showed that the quaking-induced conversion (QuIC) assay can detect sub-femtogram levels of PrPres in scrapie-infected hamster brain tissue and distinguish cerebral spinal fluid (CSF) samples from normal and scrapie-infected hamsters. During FY2009, we adapted the QuIC reaction to prion diseases of medical and agricultural interest: human variant Creutzfeldt-Jakob disease (vCJD) and sheep scrapie. Prion-positive and negative brain homogenate samples from humans and sheep were discriminated within 1-2 days with a sensitivity of 10-100 femtograms PrPres. More importantly, in as little as 32h we were able to distinguish CSF samples from scrapie-infected and uninfected sheep. This is the first time that PrPres is detected in scrapie positive derived CSF. This new method enables the relatively rapid and sensitive detection of human CJD and sheep scrapie PrPres and may facilitate the development of practical preclinical diagnostic tests.